Project Summary Mouse models have served as a powerful tool in immunology research for decades, providing incredible insight into our understanding of the immune system. However, in many cases, observations in mice do not translate to humans. Constant microbial exposure from the environment generates in humans an expansive and diverse memory T cell population, including tissue resident memory T cells (TRM). Skin TRM play critical roles in human skin health and disease. Standard laboratory mice maintained in specific pathogen free (SPF) conditions lack a true memory T cell population, including skin TRM, limiting their utility in the study of skin diseases where skin TRM may be causal or contributory. Moreover, we now know that the microbiome greatly influences immunity and inflammation, yet SPF mice have a very restricted microbiome. Recent ground-breaking work demonstrated that laboratory mice co-housed with pet shop mice acquire a robust and diverse memory T cell population akin to memory T cell populations in humans, including skin TRM. Moreover, data suggested that regulatory T cells, memory B cells and innate immune cells were also altered in the co-housed “dirty” mice. Diverse microbial exposure drove this dramatic shift in immune cell populations. Dirty mice could have a potentially profound impact on the entire field of skin immunology, by serving as a more human-like mouse model for translational research. As a first step though, the skin immune system of dirty mice must be well-characterized, and these mice tested in models of immune-mediated skin disease. In Aim 1, we will thoroughly characterize the skin immune system in dirty versus clean control mice of different strains, sex, skin site, and as a consequence of varying microbial exposure. Results will therefore be broadly applicable to the skin immunology community. We will use both novel and traditional techniques including single cell RNA sequencing, bulk RNA sequencing, high throughput TCR sequencing, high parameter flow cytometry and tissue immunofluorescence staining with microscopy. In Aim 2, we will test dirty versus clean mice in innovative models of two immune-mediated skin diseases, graft-versus-host-disease and drug hypersensitivity reaction. Ample human data support a role for memory T cells, particularly skin TRM in both diseases but lack of a mouse model containing these cells has to- date precluded mechanistic interrogation. We hypothesize that dirty mice will superiorly model these two human skin diseases and will provide a novel avenue for investigating skin TRM and microbiome in disease pathogenesis going forward. In sum, this innovative work is feasible, generalizable, and potentially transformative to the field of skin immunology.